alcoholic fermentation in multicellular organisms895 invited perspectives in physiological zoology...

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Alcoholic fermentation in multicellular organismsvan Waarde, Aren

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895

Invited Perspectives in Physiological Zoology

Alcoholic Fermentation in Multicellular Organisms Aren van Waarde* Department of Biology (Animal Physiology), Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands

Accepted 11/27/90

Abstract 1. Ethanol is an end product of anaerobic metabolism in a surprisingly large va-

riety of multicellular organisms. These include Angiosperms, Platyhelminthes, As-

chelminthes, Acanthocephala, Arthropoda, and Vertebrata. 2. Ethanol formation proceeds in two steps (via pyruvate decarboxylase and alcohol dehydrogenase). 3. Pyruvate decarboxylase of plants is located in the cytosol, whereas that of animals is intramitochondrial and probably part of the pyruvate dehydrogenase complex. 4. Alcohol dehydrogenases offacultative anaerobes are NAD- rather than NADP-dependent, with the enzyme in the parasitic worm Moliniformis dubius as the only known exception. 5. Regulation at the pyruvate branch point in

plants seems to involve three different mechanisms: (i) increases ofpyruvate de-

carboxylase and alcohol dehydrogenase activity due to increased gene expression, (ii) control ofpyruvate decarboxylase by the intracellular pH and the cytoplasmic NADH/NAD+ ratio, and (iii) inhibition of lactate dehydrogenase by ATP at low pH values. There is no evidence for increased expression of the pyruvate decarboxylase and alcohol dehydrogenase genes of animals during anoxia. Here, pyruvate decarboxylase is controlled by the intracellularpH and the mitochon- drialphosphorylation potential, whereas the ethanol/lactate production ratio is

dependent on the magnitude of the glycolytic flux. 6. The ethanol route has significant advantages (minimal acidosis, avoidance of osmotic problems, and end-

product inhibition of the glycolytic chain) but also has disadvantages (relatively low energy yield, loss of carbobydrate carbon). This may explain why only a few animals produce ethanol during anoxia, in contrast to plants, which generally respond to reduced 02 availability by alcoholic fermentation.

Introduction

Since the days of Pasteur, it has been common knowledge that yeasts produce ethanol under anoxic conditions. However, few people are aware of the fact that many higher organisms follow a similar strategy for anoxic survival.

* Present address: Division of Cardiology, Thorax Center, Academical Hospital, University of Groningen, P.O. Box 30.001, 9700RB Groningen, The Netherlands.

Physiological Zoology 64(4):895-920. 1991. 1 1991 by The University of Chicago. All rights reserved. 0031-935X/91/6404-9080$02.00



896 A. van Waarde

The present article gives an overview of recent data on ethanol production during facultative anaerobiosis. The subject matter is divided into four different topics: (i) Which species excrete ethanol and what is the quantitative importance of ethanol as an anaerobic end product? (ii) By what mechanism is ethanol produced (enzymatic reactions, subcellular distribution)? (iii) How is metabolism at the pyruvate branch point controlled? (Since at least two enzymes, lactate dehydrogenase and pyruvate decarboxylase, compete for a common substrate, some form of control seems necessary.) (iv) What are the advantages and disadvantages of the ethanol route? Consideration of these may help to explain why alcoholic fermentation is ubiquitous in

plants but relatively rare among animals.

Distribution of the Ethanol Pathway

Plants. Angiosperms may experience conditions of limited oxygen avail-

ability during all phases of their life cycle. Many seeds have a testa which is hardly permeable to oxygen (Crawford 1977, 1978; Cossins 1978). The

developing embryo is therefore subjected to a period of anoxia between imbibition and the emergence of the radicle. This period is characterized

by relatively high activities of glycolytic enzymes and by accumulation of ethanol. Leguminous plants like chick peas (Cicer arietinum; Aldasoro and Nicolas 1980), green peas (Pisum sativum; Cossins et al. 1968; Koll6ffel 1968; Suzuki and Kyuwa 1972; Leblova, Sinecka, and Vanickova 1974; Leblova 1978a, 1978b), beans (Phaseolus vulgaris; Doireau 1971; Leblova 1978b), soybeans (Glycine max; Leblova 1978b), or lentils (Lens esculenta; Leblova

1978a, 1978b), and grains like barley (Hordeum vulgare; Duffus 1968), maize (Zea mays; Leblova 1978b), and rye (Secale cereale; Leblova 1978a) produce ethanol during early development. After the testa has been ruptured, oxygen can enter and the seedling switches to aerobic respiration.

Species that grow in marshlands often have to germinate under water.

Especially in a hot climate, dissolved oxygen concentrations near the bottom can be very low. Rice (Oryza sativa; Taylor 1942; Avadhani et al. 1978; Bertani, Brambilla, and Menegus 1980; Cobb and Kennedy 1987), wild rice

(Zizania aquatica; Campiranon and Koukkari 1977), and barnyard grass (Echinochloa crus-galli; Rumpho and Kennedy 1981, 1983; Cobb and Ken-

nedy 1987) are plants that do germinate in the absence of oxygen and emerge from deep water. Ethanol is the main metabolic end product in these species under anoxic conditions (Taylor 1942; Phillips 1947; App and Meiss 1958; John and Greenway 1976; Campiranon and Koukkari 1977; Avadhani et al. 1978; Bertani et al. 1980; Cobb and Kennedy 1987). During anaerobic germination, shoots are formed but root development is suppressed. As soon



Alcoholic Fermentation in Multicellular Organisms 897

as the shoot reaches an aerobic zone, oxygen is transported downward and root growth starts (Cobb and Kennedy 1987).

The meristematic zone of roots (i.e., the apex of the root that is very actively growing) seems to experience a permanent functional anaerobiosis. Even under well-aerated conditions, the supply of oxygen to this tissue is less than its oxygen demand (Crawford 1978). Consequently, part of its

energy is produced by anaerobic pathways. Pea root tips contain high concentrations of ethanol even in an aerobic environment (Ramshorn 1957; Betz 1958; Cossins 1978). Oxygen diffusion into ripening fruits also cannot

keep pace with oxygen demand. Hypoxic and hypercapnic conditions prevail in the deep tissues of apples, pears, and bananas, which may sometimes lead to accumulation of ethanol (Zemlianukhin and Ivanov 1978).

The roots of many plants endure environmental oxygen deficiency during flooding, which can be a seasonally occurring phenomenon. The water table tends to rise during winter, and in spring it takes time to return to its summer level. At the beginning of the growth season, the deeper-lying roots of trees may thus be temporarily deprived of oxygen (Crawford 1978). Rhizomes of marsh plants, like the sea rush (Scirpus maritimus), bulrush (Schoeno- plectus lacustris), reed-mace (Typha angustifolia), and reed sweet-grass (Glyceria maxima) are adapted to overwintering in anaerobic mud (Craw- ford 1982; Monk and Brindle 1982). Ethanol is usually the major metabolic end product of roots in flooded soils (Laing 1940; Crawford 1967; Wignarajah, Greenway, and John 1976; Chirkova 1978; Smith and ap Rees 1979b; Men- delssohn, McKee, and Patrick 1981; Monk and Brindle 1982; Jackson and Drew 1984), but there are exceptions to this rule. Roots of alders (Alnus incana) produce glycerol, and those of birch trees (Betula pubescens) malate upon flooding (Crawford 1972). The anaerobic end products are transported upward and excreted (ethanol evaporates through branch len- ticels and leaf stomata; Kenefick 1962; Fulton and Erickson 1964; Chirkova 1978; Hook 1984) or assimilated into macromolecules (Cossins and Beevers 1963; Cossins 1978; Crawford 1978).

Animals. Those animal species in which ethanol production has been con- clusively demonstrated are listed in table 1. It has been suggested that a Chondrostean fish, the lake sturgeon (Acipenserfulvescens), is also capable of ethanol production (Singer, Mahadevappa, and Ballantyne 1990) since the animals (1) show a considerable resistance to anoxia, (2) do not develop an oxygen debt, and (3) possess low levels of lactate dehydrogenase in their tissues. However, until it has been proven that alcohol dehydrogenase is present and ethanol is excreted by anoxic sturgeons, the evidence must be considered only circumstantial.



TABLE

1

Distribution

of the

ethanol

pathway

among

different

systematic

groups

When

Ethanol

Other

End

Products

of

Species

Producing

Ethanol

Is Produced

Importance

of Ethanol

Anaerobic

Metabolism

Angiosperms: Virtually

all species

examined"

Germination

of seeds

with

an 02-

Usually

main

end

product

Lactate,

succinate,

shikimate,

impermeable

testa

malate,

glycerol,

etc.

Germination

under

water;

fast-

growing

root

tip;

ripening

fruit;

flooding

of the

soil;

hibernation

in anaerobic

mud

Platyhelminthes

(Cestoda):

Echinococcus

granulosush

...

Normal

life

in the

intestine

of the

Minor

end

product

Lactate,

acetate,

succinate

host

(<2%

of carbohydrate

metabolism)

Taenia

taeniaeformisc

......

See

above

See

above

Lactate,

acetate,

succinate

Aschelminthes

(Nematoda):

Aphelenchus

avenae"

........

Flooding

of the

soil?

Major

end

product

Lactate,

succinate

(50%-80%

of glycogen

consumed)

Caenorhabditis

specd

.......

See

above

See

above

Acetate,

unknown

4-carbon

alcohol

Panagrellus

redivivuse

.....

See

above

See

above

Glycerol,

lactate,

alanine,

propionate,

acetate,

acetoin

Acanthocephala: Moliniformis

dubius

........

Normal

life

in the

upper

small

Major

end

product

of

Lactate,

succinate,

acetate,

butyrate

intestine

of the

rat

glycogen

catabolism

8



Arthropoda

(Collembola):

Diverse

species

of springtails

living

on the

banks

of

freshwater

streams9

.......

Oxygen

depletion

in the

ambient

Probably

major

end

Unknown

water

product

(but

small

size

of animals

makes

precise

origin

of ethanol

difficult

to localize)

Arthropoda

(Insecta):

Chironomus

thummi

thummih

Oxygen

depletion

in the

mud

of

Major

end

product

Alanine,

lactate,

succinate

shallow,

eutrophic

waters

(>80%

of net

glycogen

utilization)

in mosquito

larvae

Chironomus

tentansi

.......

See

above

See

above

Vertebrata

(Pisces):

Carassius

auratus'

.........

Oxygen

depletion

in the

ambient

Major

end

product

Lactate

water

(>65%

of net

glycogen

utilization) Produced

in the

myotomal

muscles

Carassius

carassiusk

........

Hibernation

in small

lakes

with

See

above

Lactate

anoxic

conditions

in winter

Rhodeus

amarus'

..........

Oxygen

depletion

in the

ambient

See

above

Lactate

water

(maybe

during

larval

stage

when

animals

live

in the

mantle

cavity

of freshwater

mussels)

Note.

The

sources

for

the

information

in the

table

are:

aSee

text;

bAgosin

1957;

Cvon

Brand

and

Bowman

1961;

dCooper

and

van

Gundy

1971;

eButterworth

and

Barrett

1985;

'Crompton

and

Ward

1967;

Ward

and

Crompton

1969;

K6rting

and

Fairbairn

1972;

9E.

Zebe,

personal

communication,

1990;

hWilps

and

Zebe

1976;

Redecker

and

Zebe

1988;

'Harms

1972;

'Shoubridge

1980;

Shoubridge

and

Hochachka

1980;

kJohnston

and

Bernard

1983;

Holopainen

and

Hyv~irinen

1985;

'Wissing

1986;

Wissing

and

Zebe

1988.

8



900 A. van Waarde

Few people have searched for the presence of the ethanol pathway in

closely related species, although such information might be very interesting from a phylogenetic point of view. Wissing (1986) has examined a large number of fish species to see whether their myotomal muscles possess a

special alcohol dehydrogenase (geared toward ethanol formation). His

(negative) findings and those of other authors are summarized in the Ap- pendix. At present it is not known whether "nonproducers" that are closely related to ethanol-producing species lack the gene coding for muscle alcohol

dehydrogenase (ADH) or fail to express this gene. The answer to this ques- tion may throw light on the origin of the ethanol route.

The Mechanism of Ethanol Formation

Microorganisms

Alcoholic fermentation is well characterized in microorganisms, where ethanol is produced by two different pathways (Wilps and Sch6ttler 1980; see fig. 1). Yeasts catabolize pyruvate to acetaldehyde by a cytosolic decar-

boxylase (PDC). Acetaldehyde is then converted to ethanol by alcohol

dehydrogenase. Redox balance is maintained because the glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and ADH reactions are stoichio-

metrically coupled (Stryer 1988). Bacteria produce ethanol from pyruvate by the concerted actions of pyruvate dehydrogenase (PDH), thiokinase (TK), aldehyde dehydrogenase (AldDH), and ADH (Doelle 1975; Thauer, Jun- germann, and Decker 1977). The metabolic scheme of figure 1 is an over-

simplification, for acetyl-CoA can also be transformed to acetate via acetyl- phosphate (Doelle 1975; Shoubridge 1980), though this does not affect the overall stoichiometry of the pathway.

Pyruvate Decarboxylation in Multicellular Organisms

The mechanism of ethanol formation in plants is very similar to that of yeast cells. Ethanol is formed by PDC and ADH, which are both soluble and

located in the cytoplasm (Davies, Grego, and Kenworthy 1974; Laszlo and

St. Lawrence 1983). Mitochondria are not required for alcoholic fermentation of glucose; therefore the process can be studied in a high-speed supernatant (Davies et al. 1974).

In contrast, conversion of pyruvate to acetaldehyde is intramitochondrial in all ethanol-producing animals that have been examined (Wilps and Schdttler 1980; Mourik et al. 1982; Barrett and Butterworth 1984). Acetal-

dehyde diffuses to the cytoplasm and is there reduced to ethanol. The fer-




Glucose

Pyruvate

NAD

coA

p

NADH -------- PDC PDH

Acetaldehyde Acetyl-coA

NADH TK ADP

NAD -------- coA ATP

Ethanol Acetate

NADH-- AldDH NAD

Acetaldehyde

ADH NADH

ADH NAD

Ethanol

Fig. 1. Metabolic pathways for ethanol formation in yeasts (left) and bacteria (right). See the text for an explanation.

mentation process involves cooperation of cytosol and mitochondria, unlike the situation in yeast.

In theory, mitochondrial decarboxylation of pyruvate can take place in one step or be a multistep process involving the concerted actions of PDH, TK, and AldDH (see fig. 1). The latter route was at first favored (Shoubridge 1980; Shoubridge and Hochachka 1981) because of an extra mole of ATP obtainable at the thiokinase step and the following lines of evidence: (1) injected 14C-acetate is rapidly oxidized to 14C02 by normoxic goldfish, so a thiokinase must be present; and (2) anoxic fish produce 14C-ethanol from

injected 14C-acetate.

Subsequently, it has become clear that the thiokinase route is relatively unimportant. (a) In the step leading from acetyl-CoA to acetate, acetyl-CoA hydrolase (EC 3.1.2.1) could well outcompete acetyl-CoA synthetase (EC



902 A. van Waarde

6.2.1.1, a thiokinase); in such an event, there would be no energetic advan-

tage to ethanol formation. (b) Although acetyl CoA-synthetase is kinetically and thermodynamically readily reversible, aerobic operation of a thiokinase in the direction of acetyl-CoA is no proof of its anaerobic operation in the

opposite direction. (c) Although "4C-ethanol can be produced from injected 14C-acetate under anoxic conditions, 14C-lactate is a much better precursor (Shoubridge 1980; Shoubridge and Hochachka 1981). This suggests that acetate is not an obligate intermediate in the pathway between pyruvate and ethanol. (d) The enzyme aldehyde dehydrogenase is virtually absent in Carassius muscle (Nilsson 1988, 1990) and in mitochondria from Chi- ronomus larvae (Wilps and Sch6ttler 1980). Acetate can therefore not be

hydrogenated in situ. (e) Permeabilized mitochondria of Chironomus do not convert acetyl-CoA to ethanol (Wilps and Sch6ttler 1980). In a recon- stituted system containing intact mitochondria, ADH, NADH, and pyruvate, arsenite inhibits acetate formation, but ethanol production is unaffected

(Wilps and Sch6ttler 1980). This proves that acetate and ethanol are syn- thesized by different pathways. (f) The reactions leading from acetate to ethanol are thermodynamically uphill. Net acetate-to-ethanol conversion therefore would only be expected at very high levels of acetate or very low levels of ethanol. Since acetate concentrations in the muscle of anoxic goldfish are low and independent of oxygen availability (van den Thillart, Kes-

beke, and van Waarde 1976), acetate is probably not the obligate precursor of ethanol. (g) Barrett and Butterworth (1984) have partially purified the mitochondrial pyruvate decarboxylase of Panagrellus redivivus. Pyruvate decarboxylase activity is localized in the mitochondrial membrane fraction, but it can be solubilized by sonication or freeze/thawing and purified by isoelectric precipitation at pH 5.5. Since PDC and PDH show the same subcellular distribution and a constant activity ratio during purification, the authors conclude that PDC is a single enzyme and probably a part of the PDH complex.

For the reasons stated above, it appears that anaerobic decarboxylation of pyruvate by animal mitochondria is a one-step reaction. However, elu- cidation of the precise biochemical mechanism of acetaldehyde formation seems in order. It would be very interesting to isolate pyruvate dehydrogenase from red muscle of crucian carp or goldfish. Does the protein that converts pyruvate to acetaldehyde copurify with the PDH complex? Is PDH of ethanol-producing fish different from the enzyme in closely related species that do not produce ethanol? In case such purification may prove difficult or impossible, intact animals or isolated mitochondria could be treated with activators (dichloroacetate, lipoic acid) or inhibitors (arsenite) of pyruvate dehydrogenase to study their effects on pyruvate metabolism under aerobic and anoxic conditions.




If the pyruvate decarboxylase of ethanol-producing animals is indeed part of the pyruvate dehydrogenase complex, as proposed by Mourik et al. (1982) and suggested by the experimental evidence of Barrett and Butterworth (1984), acetaldehyde formation may proceed by the mechanism illustrated in figure 2. Reversible dissociation of bound enzymes underlies the metabolic depression that occurs in many animals during facultative anaerobiosis

H E3-FAD

-

---. . +,C-S ,-- R-I-S NADH,H- 'C-C--E1

6

CH3 NAD

E3-FADH2 1 H3C-C-COOH E2

S- -- S-S 5

COOH

E3FAD,'-' H3C-C-OH

C- SH SH R C C1-E1

CH3 0 002 H3C-C-ScoA

2 C2 coASH

H S E SH

H3C- C- OH CSOH

I1

E2 C-CH3 R NC-S2 3 11

CH3

H3C-CX-H H

6C-S ,CCC-- E1

C H3

Fig. 2. Hypothetical mechanism of acetaldehyde formation by the mitochondrial pyruvate dehydrogenase complex. During normoxia, tight coupling ofpyruvate decarboxylase (E,), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3) causes reactions 3-6 to proceed. Anoxia induces dissociation of the complex, enabling independent operation ofpyruvate decarboxylase. Reactions 1 and 2 are now followed by reaction 7, and decarboxylation ofpyruvate leads to the re- lease of acetaldehbyde.



904 A. van Waarde

(Plaxton and Storey 1986; Guderley, Jean, and Blouin 1989; Lazou, Michae- lidis, and Beis 1989). Pyruvate decarboxylase is located on the outside of the pyruvate dehydrogenase complex (Lehninger 1970), and it may be re- leased in ethanol-producing species during anoxia, so that pyruvate de-

carboxylation becomes independent of the operation of the electron trans-

port chain.

Alcohol Dehydrogenase in Multicellular Organisms

According to Chirkova (1978), ADH from the roots of a flooding-resistant plant (the willow Salix alba) is specially adapted for ethanol oxidation, whereas ADH from a flooding-intolerant plant (the poplar, Populuspetrows- kiana) has ideal properties for acetaldehyde reduction. It is doubtful whether these findings can be generalized. Purified enzymes from 13 different species with varying tolerance to flooding all show the greatest activity in the reducing direction (Davies et al. 1973; Brown, Marshall, and Munday 1976; John and Greenway 1976; Leblova 1978a).

Most alcohol dehydrogenases from ethanol-producing organisms are NAD- rather than NADP-dependent. These include the enzyme from wild rice

(Campiranon and Koukkari 1977), potato (Davies et al. 1973), barley (Duffus 1968), bean, lentil, pea, soybean, maize, cucumber, wheat, rice, rape, sun- flower (Leblova 1978a), and goldfish (Mourik et al. 1982), but there exists an exception to this rule. The parasitic worm Moliniformis dubius has no alcohol:NAD oxidoreductase (EC 1.1.1.1) and large amounts of alcohol: NADP oxidoreductase (EC 1.1.1.2, K6rting and Fairbairn 1972). This ex-

ception is interesting since a stoichiometric coupling of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ADH is normally required to maintain redox balance (Stryer 1988).

Interorgan Lactate/Ethanol Shuttles in Freshwater Fish

Cyprinids possess two isozymes of alcohol dehydrogenase with different

catalytic properties. Hepatopancreas contains the normal vertebrate isozyme which has a high affinity for ethanol (Mourik et al. 1982). This protein is

supposed to be involved in the oxidation of ethanol from the diet. The lateral red and epaxial white muscles have a peculiar isozyme with low affinity for ethanol, which is involved in alcoholic fermentation (Mourik et al. 1982). ADH activity in heart muscle and brain is below the limit of detection; these tissues are only capable of lactate glycolysis (Mourik et al. 1982).

Tracer experiments (Shoubridge 1980; Shoubridge and Hochachka 1983) and measurements of lactate gradients over the sarcolemma of the myotomal




muscles (van Waarde, van den Thillart, and Dobbe 1982) have provided evidence that lactate produced in peripheral tissues of anoxic goldfish is taken up by the myotome, where it is decarboxylated. The ultimate fate of blood lactate is excretion in the ambient water as alcohol and CO2. When mitochondria isolated from goldfish red muscle are incubated with lactate, NAD, LDH, and ADH, lactate is quantitatively converted to ethanol (G. van den Thillart and J. Warnaar, unpublished data; G. van den Thillart and M.

Verhagen, unpublished data). Mitochondrial abundance in a tissue can be assessed by measurement of

the marker enzyme succinate oxidase. Van den Thillart (1977) found 10- 18 U/g in red muscle and 1.5-1.6 U/g in white muscle of goldfish. If we assume PDC activity is proportional to mitochondrial abundance, the maximal rate of alcohol production by white fibers is only 10%-15% of that in red muscle, but owing to its large mass, white muscle may still be a quantitatively important ethanol-producing organ in the fish body.

A closer examination of Carassius gills may produce interesting results. Ekberg (1956) has observed a large anaerobic CO2 production in isolated gills of goldfish and crucian carp, which is not due to a shift of the bicarbonate equilibrium by tissue acidification and may suggest the presence of pyruvate decarboxylase. Localization of PDC and ADH in the gill could be functional since ethanol would be produced in the organ by which it is excreted.

Regulation of the Ethanol Route by Enzyme Induction

Plants. Angiosperms generally show an increase of ADH activity in response to flooding (Crawford 1967; Francis, Devitt, and Steele 1974; Wignarajah et al. 1976; Mendelssohn et al. 1981; Jenkin and ap Rees 1983; Rowland and Strommer 1986) or oxygen deprivation (App and Meis 1958; Hageman and Flesher 1960; Kollikffel 1968; Leblova et al. 1969; Wignarajah and Greenway 1976; Wignarajah et al. 1976; Campiranon and Koukkari 1977; Smith and ap Rees 1979a; Bertani et al. 1980; Rumpho and Kennedy 1981; Monk and Brandle 1982; Jenkin and ap Rees 1983; Hanson, Jacobsen, and Zwar 1984; Ricard et al. 1986; Johnson, Cobb, and Drew 1989). These changes roughly correspond to increases in ethanol production. The rise of ADH activity upon flooding or anoxic exposure is due to increased gene expression, elevated levels of mRNA, and enhanced protein synthesis (John and Green- way 1976; Sachs and Freeling 1978; Ferl, Brennan, and Schwartz 1980; Han- son et al. 1984; Ricard et al. 1986; Rowland and Strommer 1986). The ADH isozymes that are induced by oxygen deficiency have a 3-6-fold higher affinity for NADH than the normoxic isozyme, so their synthesis facilitates alcoholic fermentation of glucose (Mayne and Lea 1985). The inducer substance may



906 A. van Waarde

either be ethanol (App and Meis 1958; Koll6ffel 1968) or acetaldehyde (Hageman and Flesher 1960; Crawford and McManmon 1968; Koll6ffel 1968), depending on the species.

The capacity of plants for pyruvate decarboxylation also increases upon flooding (Wignarajah et al. 1976) and exposure to anoxia (John and Green-

way 1976; Wignarajah and Greenway 1976; Wignarajah et al. 1976; Laszlo and St. Lawrence 1983). The rise of enzyme activity is caused by de novo

synthesis of the PDC polypeptide (John and Greenway 1976; Laszlo and St. Lawrence 1983). Absolute activities of PDC are 6-15 times lower than those of ADH and closely approximate the in vivo rates of alcoholic fermentation

(John and Greenway 1976; Wignarajah and Greenway 1976). The reaction

catalyzed by PDC is therefore considered the rate-limiting step in the ethanol

pathway. The presence of excess ADH is probably essential to prevent accumulation of acetaldehyde, a highly reactive and toxic compound (Walia and Lamon 1989).

Although increases of ADH and PDC correspond to increases in ethanol

production under anoxic conditions, such a correlation is not observed dur-

ing aerobic recovery. Elevated activities of the two enzymes persist for several

days, but ethanol production stops immediately upon reoxygenation (John and Greenway 1976). This indicates the ethanol pathway is under fine control

by other mechanisms, such as the redox state, allosteric effectors, or covalent modification.

Animals. ADH activity in the muscle of normoxic goldfish is high, and the amount of enzyme is not altered by anoxic exposure (van Waarde et al. 1990a). In the nematodes Aphelenchus and Caenorbabditis, ADH activity is likewise independent of oxygen availability (Cooper and van Gundy 1971). Induction of ADH as observed in plants seems therefore to be absent in these animal species.

PDC activity in Moliniformis dubius is much (>16 times) lower than that of ADH (Kirting and Fairbairn 1972), indicating that the decarboxylase is

limiting the rate of conversion of pyruvate to ethanol.

Regulation at the Pyruvate Branch Point

Plants. Davies and co-workers (Davies et al. 1974; Davies 1980) examined the factors controlling lactate and ethanol production in cell-free extracts prepared from pea (Pisum sativum) seeds and parsnip (Pastinaca sativa) roots. They showed that PDC has an acid pH optimum, whereas LDH per- forms optimally in the alkaline range. Nucleotides (ATP, ADP, AMP) do not affect PDC, in contrast to LDH, which is inhibited by ATP, especially at low pH values. At the onset of anoxic exposure, the NADH/NAD+ ratio increases,




pHi is still >7, and LDH is not inhibited by ATP, so lactate accumulates and the pH falls. The resulting acidosis activates PDC and initiates competition for pyruvate. The ratio between lactate production and ethanol formation will be determined by (i) the pyruvate concentration, (ii) the intracellular

pH, and (iii) the concentration of ATP. The LDH/ADH activity ratio drops when the pyruvate concentration increases or the pH, falls. When the intracellular pH reaches 6.9, inhibition of LDH by ATP becomes very important and the production of lactate virtually stops. Ethanol then becomes the

major anaerobic end product. John and Greenway (1976) reported allosteric activation of PDC by NADH and inhibition by NAD+. The immediate stop of ethanol production upon reoxygenation is therefore probably due to removal of NADH by the electron-transport chain. The disappearance of NADH causes inhibition of PDC and ADH, thus effectively blocking the ethanol pathway.

Roberts et al. (1984a) examined the ethanol pathway in vivo by measuring NADH (fluorescence), lactate and ethanol production ('3C-nuclear magnetic resonance [NMR] spectroscopy), intracellular pH (31P-NMR s1ectroscopy), and ATP concentration (31P-NMR spectroscopy) in intact maize root tips during hypoxia. The model of Davies et al. (1974) provides an accurate

description of the in vivo situation. Hypoxia causes a rapid (<2 min) increase of NADH, followed by a transient (ca. 35 min) accumulation of lactate. After 35 min, pHi and lactate concentrations stabilize due to activation of the ethanol route. These observations lead to the hypothesis that H+ is the signal triggering ethanol production, the pH drop being provided by lactate glycolysis. To test this hypothesis directly, the pHi of the root tips was manip- ulated prior to anoxic exposure by addition of various amounts of acetic acid (0-5 mM) to the perfusion medium. The lag time between the onset of hypoxia and the start of ethanol production can indeed by abolished by lowering the cytoplasmic pH under a threshold value of 6.9. Viability of the root tips and the eventual kinetics of ethanol production are not affected by acid treatment, so acetic acid is not toxic at the concentrations used in this study.

Animals. Pyruvate decarboxylation by intact mitochondria of Panagrellus redivivus is inhibited by ATP (strong), NAD+ (weak), and acetyl-CoA (mod- erate) but unaffected by ADP, AMP, cAMP, NADH, NADP+, and NADPH (Barrett and Butterworth 1984). Van den Thillart and Verhagen (unpublished data) have observed that decarboxylation of pyruvate in muscle mitochondria of goldfish is modulated by the ATP/Pi ratio. When 10 mM Pi, 0.1 mM ad- enylylimidodiphosphate (AMPPNP, an ATPase inhibitor), and no ATP are present, PDC shows maximal activity. In the presence of 10 mM ATP, 0.1 mM AMPPNP, and no Pi, the reaction rate is reduced to 15% of the maximal



908 A. van Waarde

value. Changes in the pH of the incubation medium have little influence on the rate by which anoxic mitochondria decarboxylate pyruvate in the presence of 10 mM Pi, but a decline of the pH from 7.4 to 6.9 abolishes the inhibition of PDC by ATP (10 mM). Mourik et al. (1982) have reported that anaerobic pyruvate decarboxylation in muscle mitochondria of goldfish is stimulated by ADP.

In all animal species that have been examined, lactate and alanine ac- cumulate initially but not during the later stages of the anoxic period, when ethanol becomes the major end product (Cooper and van Gundy 1971; van den Thillart et al. 1976; Wilps and Zebe 1976; van den Thillart and van Waarde 1990). Fish do not produce ethanol during normoxia (van den Thil- lart, van Berge Henegouwen, and Kesbeke 1983; van Waversveld, Addink, and van den Thillart 1989) or physical exercise (Wissing and Zebe 1988) but only during environmental hypoxia/anoxia (van den Thillart et al. 1983; Wissing and Zebe 1988; van Waversveld et al. 1989). The ethanol/lactate production ratio during anoxia seems dependent on the glycolytic flux, rel-

atively more lactate being produced when the flux increases (Van Waarde et al. 1990a; see table 2).

These observations suggest the following control at the pyruvate branch

point. At the onset of anoxia, the NADH/NAD+ ratio shifts to the more

TABLE 2

Dependence of the lactate/ethanol production ratio in anoxic goldfish on the glycolytic flux

200C-acclimated 5SC-acclimated

Energy demand (CO2 production)a ............ .. 282 83

Ethanol excretion during steady state" ............ 289 68

Lactate formation during steady state ........... ....... 302 27

Ethanol/lactate production ratioc .................. .95 2.4

Note. Values expressed as Cgmol * 100 g-' o

h-1 a Data from van den Thillart et al. 1983. b Estimated from the pH decline in the epaxial white muscle (measured by 31P-NMR spectroscopy), assuming a buffering capacity of 30 meq H+/pH unit and negligible leak-out of protons during anoxia (data from van Waarde et al. 1990a). c Assuming a correlation between myotomal and whole-body rates of glycolysis.




reduced state, facilitating glycolysis and causing a net drop in the intracellular pH. Inorganic phosphate (Pi) accumulates because anaerobic energy metabolism cannot maintain the free ADP concentration near the normoxic level that is mainly determined by oxidative phosphorylation:

ATP -- ADP + Pi (cellular energy demand),

phosphocreatine + ADP * creatine + ATP (creatine kinase),

net: phosphocreatine -+ creatine + Pi (appearance of Pi).

The declines of pHi and the ATP/ADP - Pi ratio activate PDC and initiate

competition for pyruvate. Ethanol concentrations are kept low by excretion, whereas lactate accumulates in situ. The AGo value for conversion of pyruvate to ethanol and CO2 is -8.8 kcal

1 mol-', whereas that of the lactate dehydrogenase reaction is -6 kcal - mol-'. Ethanol formation is therefore ther-

modynamically advantageous over lactate glycolysis, especially in the presence of elevated levels of lactate. However, the maximal rate of pyruvate decarboxylation is relatively low since both the substrate (pyruvate) and product (acetaldehyde) must be transported through the mitochondrial membrane and the Vmax of PDC is at least an order of magnitude lower than that of LDH. The ratio of ethanol formation over lactate production is therefore critically dependent on the glycolytic flux. At very high flux (intense exercise), virtually all carbohydrate carbon is converted to lactate. At low fluxes, which occur during environmental anoxia, ethanol production becomes relatively important.

The differences between plants and animals as far as regulation at the pyruvate branch point is concerned are summarized in table 3.

Functional Trade-offs in Alcoholic Fermentation

Alcoholic fermentation of carbohydrates has at least three significant advantages over lactate glycolysis.

1. Assuming effective removal of CO2 (see below), ethanol formation does not acidify the cell, whereas coupling of Embden-Meyerhof glycolysis with cell ATPases generates two protons per mole of glucose consumed (Hochachka and Mommsen 1983; Partner 1987). Ethanol-producing organisms may thus prevent the acidosis that is normally associated with anaerobic metabolism. Every alternative pathway of glycogen degradation (to opines, alanine, malate, succinate, propionate, or acetate) produces acid equivalents (Pbrtner 1987).

2. Ethanol is a neutral end product that can be transported by passive diffusion and is easily excreted into the ambient water (Shoubridge and



910 A. van Waarde

TABLE 3 Regulation at the pyruvate branch point

Plants Animals

1. Hypoxia-induced increases 1. Appearance of PDC during anoxia of the activities of PDC (dissociation of the PDH complex)? and ADH due to increased No changes in ADH gene expression gene expression

2. Control of cytosolic PDC 2. Control of mitochondrial PDC by the

by the NADH/NAD+ ratio ATP/ADP - Pi ratio and the and the intracellular pH intracellular pH

3. Inhibition of LDH by ATP 3. Thermodynamic advantage of ethanol at low pH values formation over lactate production at

elevated levels of lactate (ethanol is excreted into the ambient water in contrast to lactate, which accumulates in situ)

4. Ethanol pathway triggered 4. Ethanol pathway triggered by by a drop of the declines of the ATP/ADP * Pi ratio intracellular pH

Hochachka 1980). The lactate anion on the contrary is highly charged and usually accumulates in situ. Species that have to survive long periods of anoxia and produce ethanol thus avoid the osmotic problems that nor-

mally arise from extensive glycogen degradation. Another well-known solution of the osmotic problem is opine formation, that is, reductive condensation of pyruvate with an amino acid, which occurs in many invertebrates.

3. Since ethanol concentrations are kept low by passive efflux, there is no product inhibition of the glycolytic chain. The period during which anoxia can be resisted is thus limited by the amount of available substrate (i.e., glycogen) rather than end-product accumulation.

In vivo 31P-NMR studies of plants (Roberts et al. 1984a, 1984b; Roberts, Andrade, and Anderson 1985) and animals (van den Thillart et al. 1989; van den Thillart and van Waarde 1990; van Waarde et al. 1990a, 1990b; see fig. 3) have provided ample evidence to support the hypothesis that the switch to alcohol production dampens metabolic acidosis and increases the period during which cells can resist anoxia. Mutant maize roots that lack alcohol dehydrogenase do not regulate cytoplasmic pH well and cannot maintain high ATP levels in the absence of oxygen (Rob-




erts et al. 1984a, 1984b). When the intracellular pH of hypoxic root tips is experimentally lowered by exposure to various amounts of carbon dioxide, survival is shortened in proportion to the pH decrease (Roberts et al. 1984b). In anoxic goldfish, there is an initial drop of pHi due to lactate glycolysis, but during the rest of the anoxic period, acid production is suppressed (van den Thillart et al. 1989; van Waarde et al. 1990a, 1990b). The dampening of acidosis coincides with the onset of ethanol

production in the myotome (van den Thillart and van Waarde 1990; van Waarde et al. 1990b). In a comparative study using different fish species, anoxic survival correlated with the ability to stabilize the intracellular

pH (van Waarde et al. 1990a).

Although the switch from lactate to ethanol as the anaerobic end product may prolong anoxic survival, it should be emphasized that this metabolic modification has its drawbacks, too.

1. Although identical to lactate glycolysis, the process has a relatively low energy yield of 2 mol ATP per mole of glucose consumed, as opposed to 4 mol ATP per mole of glucose in succinate production and 7 mol ATP

per glucose unit in propionate production (Pirtner 1987). This means that

relatively high glycolytic fluxes are required to meet a certain energy demand, and much glycogen has to be stored for long-term anoxic survival. It is not surprising that hibernating crucian carp, which rely on the ethanol

pathway, have the highest glycogen stores known in any vertebrate. Liver

glycogen concentrations reach 30% of liver dry weight during the fall, and the organ increases in size from 2% to 14% of total body weight (Holo- painen and Hyvdirinen 1985; Hyv1irinen, Holopainen, and Piironen 1985; Piironen and Holopainen 1986). Glycogen stores in the myotome also reach record highs of 3.5% on a dry-weight basis (Hyvdirinen et al. 1985). Anoxic crucian carp do not fall into torpor but remain conscious and capable of active avoidance of a net or of predators. Since the animals do not feed during winter, huge glycogen reserves are presumably necessary for glycolytic energy production. The requirement of large glycogen stores is less stringent in invertebrates since these take up food under anoxic stress (K6rting and Fairbairn 1972; Wilps and Zebe 1976; Redecker and Zebe 1988).

2. Because ethanol is excreted, the alcohol route is wasteful of carbon. Ethanol-producing animals that are exposed to long periods of anoxia will need a good food supply between anoxic events to ensure growth and re- plenishment of liver glycogen. Crucian carp inhabit small lakes and shallow ponds with a high production of invertebrates during summer. Chironomus larvae likewise occur in shallow, eutrophic waters where the availability of food is not a problem.



Carassius carassius

mM pH 15-7. 8 I TH

S1// p 15 10-7.4

15I 10- I +I

5-L-oA C

S3 6 9 12 15 18 2 3 6 9 12 15 18 21 2 0 3 6 9 12 15 18 21 24 3 6 9 12 15 1821 24

lac 1.4A 4.0 7.0 umol/g eth 02 0.5 1.6

7.5

PH

7.0

6.5 0 1 2

ANOXIA (h)

Fig. 3. Dampening of acidosis in freshwaterfish by the ethanol pathway.

Top, measurements performed on a free-swimming, cannulated crucian

carp (Carassius carassius) at 150C. Indicated are the concentrations of




Carbon loss may be one of the reasons why the ethanol route is ubiquitous in the plant kingdom but relatively rare among animals. Plants have an abundant carbon supply in the form of atmospheric CO2, whereas animals must feed to acquire substrates for gluconeogenesis. However, a shortage of essential nutrients in plants with anoxic roots may limit their capacity for CO2 fixation. Animals are exposed to "global anoxia," but in plants normally only certain organs (i.e., the roots) are deprived of oxygen. Plants thus have the opportunity to transport ethanol upward and use it for carbohydrate synthesis, while in animals the end product of fermentation is irrevers-

ibly lost. 3. Animals cannot allow ethanol or acetaldehyde to reach high con-

centrations since these compounds disturb the operation of the nervous

system. Only those species that can keep ethanol levels low by excretion

may opt for this survival strategy. The use of alcoholic fermentation also

requires efficient removal of metabolic carbon dioxide. If CO2 accumulates in situ, the major advantage of ethanol production (dampening of acidosis) disappears because hydration of CO2 generates acid equivalents. Animals that live in water are at an advantage here since both ethanol excretion and elimination of CO2 are facilitated by the aquatic environment.

The ethanol route may further be rare among animals because its advan-

tages become significant only when high glycolytic fluxes have to be maintained for long periods of time (species that remain active, do not fall into torpor, and are subjected to prolonged anaerobiosis). Conscious activity during weeks or months in an anoxic environment requires many adaptations on other levels than energy metabolism, such as recycling of neurotrans- mitters in the brain (Nilsson 1989a, 1989b). Relatively few animals may have solved all problems of this situation and thus possess the ethanol pathway. Most species have opted for a stronger suppression of the metabolic rate, which causes torpor, and an energetically more efficient form of anaerobic metabolism to ensure anoxic survival.

lactate (0) and ethanol (0) in the blood, the plasma pH (+), and the amount of ethanol excreted in the environmental water (X). Bottom, measurements performed on six restrained goldfish (Carassius auratus) at 200 C by in vivo 31P-NMR spectroscopy (pH) and enzymatic analysis of tissue samples (lactate, ethanol). Indicated are the intracellularpH (0) of white muscle (mean 1 SE), the lactate concentration in white muscle, and the ethanol concentration in the blood. Data are from van den Thil- lart and Heisler (unpublished), van den Thillart and van Waarde 1990, and van Waarde et al. 1990a.



914 A. van Waarde

Appendix

TABLE Al Related species that do not produce ethanol

Species Reference

Arthropoda (Insecta): Culexpipiens .................. Redecker and Zebe 1988

Vertebrata (Pisces): Abramus brama ................ Wissing 1986 Anguilla anguilla .............. van Waarde, van den

Thillart, and Kesbeke 1983

Barbus ticto ................... Wissing 1986 Brachydanio rerio .............. Wissing 1986 Colossoma brachyponum ........ Wissing 1986 Colossoma macropomum ........ Wissing 1986 Ctenopharyngodon idella ........ Wissing 1986 Cyprinus carpio ................ Wissing 1986 Esox lucius .................... Wissing 1986 Gasterosteus aculeatus .......... Wissing 1986 Gobio gobio .................... Wissing 1986

Hypopthalmichthys molitrix ...... Wissing 1986 Ictalurus nebulosus ............. Wissing 1986 Leucaspius delineatus ........... Wissing 1986 Leucaspius idus ................. Wissing 1986 Oreochromis mossambicus ....... van Waarde et al. 1990b Percafluviatilis ................ Wissing 1986 Poecilia reticulata .............. Wissing 1986 Rasbora chrysotaenia ........... Wissing 1986 Rutilus rutilus ................. Wissing 1986 Tinca tinca ................... W issing 1986 Xiphophorus maculatus ......... Wissing 1986

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